Generally speaking, there are two major types of immune response: the humoral response which is characterized by the production of antibodies by B lymphocytes, and the cell-mediated immune response. Antibodies are able to recognize antigens in their three dimensional form, either soluble or bound to an insoluble support such as a cell, while T cells recognize processed antigen fragments which are bound and presented by glycoproteins encoded by the major histocompatibility complex (MHC) notably MHC-I genes which are expressed at the cell surface of almost all vertebrate cells or MHC-II genes which are expressed on antigen presenting cells (APC).
A cell-mediated immune response usually necessitates the cooperation of helper T lymphocytes and effector cells. This cooperation takes place, in particular, as a result of interleukin-2 and/or various other cytokines which are secreted by helper T lymphocytes after their activation by antigenic fragments presented by APC in association with MHC-II. Cytotoxic T Lymphocytes (CTL) are activated, induced to proliferate and to exert their antigen-specific cytotoxic function upon exposure to antigenic polypeptides complexed with autologous MHC-I, co-stimulatory molecules on the surface of the APC and cytokines, often derived from helper T cells. T cell derived cytokines can also trigger and drive the proliferation and antigen processing capacity of APC as well as activation and induction of proliferation in other cells, including other T cells.
Thus, cytotoxic T lymphocytes (CTL) recognise epitopes bound to MHC class I molecules on the surface of cells. Recognition of such epitopes on the surface of target cells by CTL leads to the killing of the target cells by the CTL. The epitopes which are displayed on the cell are fragments from proteins which have been processed in the class I antigen processing pathway of the cell. In this pathway proteins (generally from the cytoplasm) are broken down in the cytoplasm into small peptides. The small peptides are then transported through the endoplasmic reticulum (where they bind to the MHC molecules) to the surface of the cell.
Said antigen presentation by MHC-I molecules has been characterized (see for example Groettrup et al., 1996, Immunology Today 17, 429-435): a full-sized protein or glycoprotein antigen is digested into shorter antigenic polypeptides (of about 7 to 13 amino acids in length). Said polypeptides are associated with MHC-I molecules and β-2 microglobulin leading to a ternary complex which is further presented on the cell surface.
It is not possible to predict which proteins will enter the antigen processing pathway, which fragments will be produced, or which fragments will bind to MHC molecules and be presented at the surface of the cell. Additionally it is not possible to predict which fragments T cells will recognise and whether the T cells which recognise the fragments will be protective.
MHC-I specificity towards antigens can vary greatly depending on the considered MHC-I molecule (HLA-A, HLA-B, . . . ) and on the allele (HLA-A2, HLA-A3, HLA-A11, . . . ) since genes encoding the MHC molecules are greatly variable between individuals among a species (reviewed in George et al., 1995, Immunology Today, 16, 209-212).
Most tumor cells express antigens at their surface which differ either qualitatively or quantitatively from the antigens present at the surface of the corresponding normal cells. These antigens are specific when they are expressed only by tumor cells. When they are present on both normal and tumor cells, these antigens are said to be associated with the tumor; in this case, they are present either in larger amounts or in a different form in the tumor cells.
It is now well known that patients suffering from a cancer may develop an immune response to their tumor. This has been revealed, in particular, by demonstrating that the serum of some patients contain anti-tumor antigen antibodies, and that their serum was capable of inhibiting the growth of cancer cells in vitro. Nevertheless, inasmuch as spontaneous tumor regressions are extremely rare, it appears that the immune response observed in vitro remains ineffective in vivo.
Hellstrom et al. (1969, Adv. Cancer Res. 12, 167-223) have shown that antigen-specific CTL can be effective mediators in a tumor-specific immune response. However, this natural immune response is not always effective enough to limit tumor growth. Although an immune response may develop against a tumor, it is not known whether it is of real benefit to the patient. Seemingly uncontrolled tumor growth would suggest that a tumor eludes the body's mechanisms of immune surveillance. Tumor-derived molecules are considered to play a significant part in modifying or diverting the immune response in favor of the tumor rather in favor of the individual.
In the light of the complexity of the immune response against tumors and the modest state of current knowledge in this field, the use of an anticancer vaccine is not obvious. Animal studies have shown that immunization using living or killed cancer cells could lead to rejection of a subsequent tumor graft, however attempts at immunization using acellular products, for example administration of the complete antigenic protein, with polypeptide fragments of such protein DNA fragment encoding all or part of tumor-associated proteins, have generally been less successful.
Recently, Toes et al. (1997, Proc. Natl. Acad. Sci. 94, 14660-14665) have developed an alternative approach based on minimal antigenic polypeptide fragments selection which might be specifically recognized by the CTL. According to said method, the minimal antigenic fragments are expressed in the host cells where they can be associated with MHC-I molecules and then be presented on the cell surface, inducing a specific immune reaction. More specifically, it has been shown that intra-cellular expression of “minigens” encoding very short epitopes (from 7 to 13 amino acids in length) can induce a cellular immune response. Moreover, Whitton et al. (1993, J. of Virology 67, 348-352) have proposed the use of a vector, called “string of beads” construct, which co-expresses several minigens and can induce a synergetic CTL immune response.
Another recent and important use for such polypeptides is in association with soluble complexes of MHC-I, β-2 microglobulin and a fluorescent or otherwise visually detectable reagent. These, so called “Tetramers” (eg, as described in Altman et al, 1996, Science, 274:94-96) can be used to identify by flow cytometry or histology, antigen specific CTL ex vivo.
MUC-1 is a glycosylated mucin polypeptide found on the apical surface of mucin-secreting epithelial cells in various tissues, including breast, lung, pancreas, stomach, ovaries, fallopian tubes, and intestine (Peat et al., 1992, Cancer Res. 52:1954-60—Ho et al., 1993, Cancer Res. 53:641-51). Malignant transformation of breast, ovary, pancreas and probably other epithelial tissues, results in over expression of MUC-1 polypeptide in tumor cells (Hareuveni et al., 1990, Eur. Journ. Biochem. 189:475-86; Layton et al., 1990, Tumor Biol. 11:274-86). In addition, abnormal glycosylation of MUC-1 polypeptide in breast, and probably other MUC-1-expressing tumour cells results in the exposure of tumor-associated antigenic epitopes on the protein core of MUC-1 (Burchell et al., 1987, Cancer Res. 47:5476-82; Devine et al., 1990, J. Tumor Marker Oncol. 5:11-26; Xing et al., 1989, Immun. Cell Biol. 67:183-95) as well as on the glycosyl side chains (Samuel et al., 1990, Cancer Res. 50:4801-8).
Monoclonal antibodies specific for these epitopes have been described which can identify more than 90% of breast and pancreatic tumors. Non major-histocompatibility-complex (MHC) restricted cytotoxic T cell responses to the MUC-1 tumor specific protein epitope by T cells from breast and pancreatic cancer patients have also been reported (Jerome et al., 1991, Cancer Res. 51:2908-16) in addition to MHC restricted, MUC-1-specific CTL (Reddish et al., 1995, Int. J. Cancer 10:817-823). Moreover, proliferation of T cells to purified MUC-1 has been seen (Keydar et al., 1989, Proc. Natl. Acad. Sci. USA 86:1362-6). These various observations suggest that MUC-1 may be an effective target antigen for active immunotherapy in breast, as well as other cancers. Hareuveni et al. (1991, Vaccine 9:618-27) expressed the MUC-1 antigen in vaccinia virus and showed that rat immunized with VV-MUC-1 rejected MUC-1-bearing tumor cells at a rate of 60-80% (Hareuveni et al., 1990, Proc. Natl. Acad. Sci. USA 87:9498-502).